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We aimed to identify the functional characteristics of the corpus luteum (CL) by color Doppler ultrasonography and changes in interferon-stimulated gene (ISG) expression in peripheral blood mononuclear cells (PBMCs) during early pregnancy in beef cows. We then aimed to use these features to establish earlier pregnancy diagnosis methods. In experiment 1, the CL size and blood flow were accessed by Doppler ultrasonography, and the PBMCs were isolated on Days 8, 12, 15, 18, 20, 22, 25, 30, 45, and 60 post-timed artificial insemination (TAI) from pregnant (n = 10) and nonpregnant cows (n = 12). The abundance of ISG (OAS1, MX1, MX2, and ISG15) transcripts was measured by quantitative PCR. Analyses of OAS1 and MX2 expression in isolated PBMCs (ISG-PBMC method) and Doppler imaging of CL (Doppler-US method) were performed to test the accuracy of these methods for the diagnosis of pregnancy on Day 20 post-TAI (n = 110; experiment 2). In experiment 1, the luteal volume and blood flow were reduced in nonpregnant cows during the first weeks post-TAI, but an evaluation of CL vascularization and size was efficient in identifying nonpregnant cows on Day 20 post-TAI. The expression of ISGs in PBMCs can be stimulated by the presence of a viable conceptus between Days 15 and 22 post-TAI, and the expression of these genes reaches a peak on Day 20. In experiment 2, the Doppler-US and ISG-PBMC methods resulted in similar specificity (85.5 and 87.7%, respectively). However, only the Doppler-US method resulted in 100% sensitivity. In conclusion, the greatest abundance of ISGs in PBMCs and a high detection of luteolysis by Doppler imaging on Day 20 post-TAI can be feasibly used for the earlier detection of nonpregnant cows in reproductive programs. The level of accuracy for our described pregnancy methods is high on Day 20 (80%-91%), but only the Doppler imaging method results in an absence of false-negative diagnoses.

Our objective was to determine whether pregnancy rates in heat-stressed dairy cattle could be enhanced by timed embryo transfer of fresh (nonfrozen) or frozen-thawed in vitro-derived embryos compared to timed insemination. Ovulation in Holstein cows was synchronized by a GnRH injection followed 7 d later by PGF2 alpha and a second treatment with GnRH 48 h later. Control cows (n = 129) were inseminated 16 h (d 0) after the second GnRH injection. On d 7, a fresh (n = 133) or frozen-thawed (n = 142) in vitro-derived embryo was transferred to cows assigned for timed embryo transfer after categorizing the corpus luteum by palpation per rectum as 3 (excellent), 2 (good or fair), 1 (poor), and 0 (nonpalpable). Response to the synchronization treatment, determined by plasma progesterone concentration (ng/ml) < or = 1.5 on d 0 and > or = 2.0 on d 7, was 76.2%. Mean plasma progesterone concentration on d 7 increased as the quality of corpus luteum improved from category 0 to 3. Concentrations of progesterone in plasma were elevated (> or = 2.0 ng/ml) at 21 d in 64.7 (fresh embryo), 40.3 (frozen embryo), and 41.4 +/- 0.1% (timed insemination) of cows, respectively. Cows that received a fresh embryo had a greater pregnancy rate at 45 to 52 d than did cows that received a frozen-thawed embryo or timed insemination (14.3 > 4.8, 4.9 +/- 2.3%). Body condition (d 0) of cows influenced the pregnancy rate and plasma progesterone concentrations. In summary, timed embryo transfer with fresh in vitro-produced embryos in heat-stressed dairy cattle improved pregnancy rate relative to timed insemination.

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The bovine pre-implantation embryo secretes bioactive molecules from early development stages, but effects on endometrial function are reported to start only after elongation. Here, we interrogated spatially defined regions of the endometrium transcriptome for responses to a day 7 embryo in vivo. We hypothesize that exposure to an embryo changes the abundance of specific transcripts in the cranial region of the pregnant uterine horn. Endometrium was collected from the uterotubal junction (UTJ), anterior (IA), medial (IM) and posterior (IP) regions of the uterine horn ipsilateral to the CL 7 days after estrus from sham-inseminated (Con) or artificially inseminated, confirmed pregnant (Preg) cows. Abundance of 86 transcripts was evaluated by qPCR using a microfluidic platform. Abundance of 12 transcripts was modulated in the Preg endometrium, including classical interferon-stimulated genes (ISG15, MX1, MX2 and OAS1Y), prostaglandin biosynthesis genes (PTGES, HPGD and AKR1C4), water channel (AQP4) and a solute transporter (SLC1A4) and this was in the UTJ and IA mainly. Additionally, for 71 transcripts, abundance varied according to region of the reproductive tract. Regulation included downregulation of genes associated with proliferation (IGF1, IGF2, IGF1R and IGF2R) and extracellular matrix remodeling (MMP14, MMP19 and MMP2) and upregulation of anti-adhesive genes (MUC1) in the cranial regions of uterine horn. Physical proximity to the embryo provides paracrine regulation of endometrial function. Embryo-independent regulation of the endometrial transcriptome may support subsequent stages of embryo development, such as elongation and implantation. We speculate that successful early embryo-dependent and -independent programming fine-tune endometrial functions that are important for maintenance of pregnancy in cattle.

This study aimed to characterize the endometrial transcriptome and functional pathways overrepresented in the endometrium of cows treated to ovulate larger (≥13 mm) versus smaller (≤12 mm) follicles. Nelore cows were presynchronized prior to receiving cloprostenol (large follicle [LF] group) or not (small follicle [SF] group), along with a progesterone (P4) device on Day (D) -10. Devices were withdrawn and cloprostenol administered 42-60 h (LF) or 30-36 h (SF) before GnRH agonist treatment (D0). Tissues were collected on D4 (experiment [Exp.] 1; n = 24) or D7 (Exp. 2; n = 60). Endometrial transcriptome was obtained by RNA-Seq, whereas proliferation and apoptosis were assessed by immunohistochemistry. Overall, LF cows developed larger follicles and corpora lutea, and produced greater amounts of estradiol (D-1, Exp. 1, SF: 0.7 ± 0.2; LF: 2.4 ± 0.2 pg/ml; D-1, Exp. 2, SF: 0.5 ± 0.1; LF: 2.3 ± 0.6 pg/ml) and P4 (D4, Exp. 1, SF: 0.8 ± 0.1; LF: 1.4 ± 0.2 ng/ml; D7, Exp. 2, SF: 2.5 ± 0.4; LF: 3.7 ± 0.4 ng/ml). Functional enrichment indicated that biosynthetic and metabolic processes were enriched in LF endometrium, whereas SF endometrium transcriptome was biased toward cell proliferation. Data also suggested reorganization of the extracellular matrix toward a proliferation-permissive phenotype in SF endometrium. LF endometrium showed an earlier onset of proliferative activity, whereas SF endometrium expressed a delayed increase in glandular epithelium proliferation. In conclusion, the periovulatory endocrine milieu regulates bovine endometrial transcriptome and seems to determine the transition from a proliferation-permissive to a biosynthetic and metabolically active endometrial phenotype, which may be associated with the preparation of an optimally receptive uterine environment.

The continuum of folliculogenesis begins in the fetal ovary with the differentiation of the oogonia and their isolation within the primordial follicles. Primordial follicle activation is an enigmatic process, whereby some follicles enter the growing pool to become primary follicles, thereby embarking on an irreversible progression towards ovulation or atresia. This process is under the coordinated regulation of factors from the oocyte itself, as well as from the somatic cells of the ovary, in particular the theca and granulosa cells, which are structural components of the follicle. These two influences provide the principal stimuli for the growth of the follicle to the late preantral or early antral stage of development. The endocrine effects of the gonadotrophins FSH and LH are essential to the continued progression of the follicle and most atresia can be attributed to the failure to receive or process the gonadotrophin signals. The peri-ovulatory state has received intensive investigation recently, demonstrating a coordinated role for gonadotrophins, steroids, epidermal growth factor family proteins and prostaglandins. Thus, a complex programme of coordinated interaction of governing elements from both germ and somatic cell sources is required for successful follicle development.